Synergistic compositions for lycopene and Vitamin E for the prevention of LDL oxidation

ABSTRACT

A synergistic mixture containing lycopene and Vitamin E and its use in the prevention of LDL oxidation. Lycopene is either synthetic or from natural sources. Furthermore the preparation of a pharmaceutical and a dietary composition for arresting the progression of atheroclerosis is described.

CROSS REFERENCE TO RELATED APPLICATION

The present application is the national stage under 35 U.S.C. 371 ofPCT/IL98/00286, filed Jun. 18, 1998.

FIELD OF THE INVENTION

The present invention concerns a composition containing lycopene andVitamin E.

This present invention more particularly relates to a synergisticmixture of lycopene and vitamin E and its use in the prevention of LDLoxidation, and preparing pharmaceutical and dietary compositions forarresting the progression of atheroclerosis.

BACKGROUND OF THE INVENTION

Atherosclerosis is the major single cause of mortality in westernsociety. It is assumed to reach up to 50% of all mortality (Davies M J,and Woolf N. Atherosclerosis: what is it and why does it occur? Br HeartJ 69:S3, 1993). In addition, it results in significant cardiacmorbidity, such as anginal syndromes, myocardial infarctions, ischemiccardiomyopathy, sudden cardiac death, cerebrovascular accidents, andperipheral vascular disease. Indisputable evidence for an associationbetween coronary heart disease (CHD) and risk factors, such as arterialhypertension, cigarette smoking and hyperlipidemia, has been derivedfrom a variety of epidemiological studies. Of all the risk factorsestablished thus far, lipid disorders play a key role in thepathogenesis of atherosclerotic vascular disease, especially of CHD.Many epidemiological and clinical trials have demonstrated the powerfulassociation between hyperlipidemia and the widespread incidence of CHD.The Framingham Heart Study (Castelli W P, Anderson K, Wilson P W., LevyD. Lipids and risk of coronary heart disease. The Framingham Study. AnnEpidemiol 2(1-2): 23-28, 1992), which has been continuous since 1984,showed that hypercholesterolemia is a major contributor to thedevelopment of CHD. The link between atherosclerosis and cholesterol hasbeen confirmed by a number of clinical trials.

Fats are insoluble in the aqueous medium of the blood. Thus, transportof the lipids triglycerides, phospholipids and cholesterol occursexclusively by way of lipid-protein complexes, the lipoproteins. Thelipoproteins are classified into 4 broad classes-chylomicrons, very lowdensity lipoprotein (VLDL), low density lipoprotein (LDL), and highdensity lipoprotein (HDL), which differ in their composition, size, andpotential atherogenicity.

Measurement of total cholesterol level in plasma reflects the sum ofcholesterol being transported in each individual lipoprotein. However,LDL and HDL are the main cholesterol carriers in plasma, and only asmall fraction of cholesterol is carried in VLDL or in the chylomicrons.Atherosclerosis is a gradual pathological process, which ischaracterized by an accumulation of lipid filled macrophages (foamcells), and smooth muscle cells resulting in lesions that thicken andharden the arterial wall. The main source for the cholesterolaccumulating in the foam cells is the circulating LDL. There is evidencefrom numerous epidemiological and clinical studies that LDL, as thecarrier of ca. 70% of the total cholesterol in plasma, are the mostpotent atherogenic lipoproteins. Their elevation carries a particularrisk, and reduction in LDL cholesterol constitutes a diminishedatherosclerotic risk. The Lipid Research Clinics Coronary PrimaryPrevention Trial (LRC-CPPT) had for the first time presented firm proofthat in man, a lowering of LDL cholesterol level reduces the rate ofmyocardial infarcaiton and infarction mortality.

Nearly all cells, including macrophages, take up exogenous cholesterolvia LDL-receptors. Increase in cell cholesterol content, however,results in down-regulation of LDL receptor number, thereby protectingcells from excessive accumulation of cholesterol by way of this pathway.It was shown that chemical or biological modification, includingoxidation of LDL, results in increased uptake of the modifiedlipoprotein by way of other cell surface receptors, termed scavengerreceptors. These receptors are present on macrophages and endothelialcells.

Oxidative modification of LDL is thought to play a causal role inatherosclerosis: (see e.g. Steinberg D., Parthasarathy S., Carew T. E.,Khoo J. C. and Witztum J. L, “Beyond cholesterol: modifications oflow-density lipoprotein that increase its atherogenicity”, N. Engl. J.Med. 1989; 320: 915-924; Haberland M. E. and Fogelman A. M., “The roleof altered lipoproteins in the pathogenesis of atherosclerosis”, Am.Heart. J. 1987; 113: 573-577; and Witztum J. L., “The oxidationhypothesis of atherosclerosis”, Lancet. 1994; 344, 793). It is believed,accordingly, that prevention of LDL oxidation by antioxidants may arrestthe progression of atherosclerosis. Aviram M. Beyond cholesterol:Modification of lipoproteins and increased atherogenicity. InAtherosclerosis, inflammation and thrombosis (G. G. Neri Serneri, G. F.Gensini, R. Abbate and D. Prisco eds)-Scientific Press-Florence, Italy,pp: 15-36, 1993.

The ability to prevent the development of atherosclerotic lesions wouldhave major implications for the public health. Thus using therapeuticagent with plural effects, such as lowering cholesterol and inhibitingoxidative modification, might have beneficial effects over otherindividual agent.

Carotenoids are colored pigments with lipophilic properties, widelydistributed in fruits and vegetables, (e.g. β-carotene in carrots andlycopene in tomatoes) and possess some antioxidant properties: (see e.g.Burton G. W., “Antioxidant action of carotenoids,” 1989; J. Nutr.119:109-111 and Krinsky N. I., “Antioxidant functions of carotenoids”,Free Radic. Biol. Med. 1989, 7: 617-635 (9-13)). Carotenoids aretransported within circulating lipoproteins, and it was postulated thatthey participate in the protection of LDL from oxidative modification.

Carotenoids consumption was shown in previous epidemiological studies tobe associated with reduced cardiovascular mortality (see e.g.: KohlmeierL. and Hasting S. B., “Epidemiologic evidence of a role of carotenoidsin cardiovascular disease prevention”, Am. J. Clin. Nutr. 1995; 62:137S-146S), although recent data did not demonstrate similar beneficialeffect (see e.g.: Hennekens C. H., Buring J. E., Manson J. E., StampferM, Rosier B., Cook N. R., Belanger C., LaMotte F., Gaziano J. M., RidkerP. M., Willett W. and Peto R., “Lack of effect of long-termsupplementation with beta carotene on the incidence of malignantneoplasms and cardiovascular disease”, N. Engl. J. Med. 1996; 334:1145-1149).

Reduced plasma lipid peroxidation was recently shown to be associatedwith increased consumption of tomatoes. Low levels of plasma carotenoidswere shown to be associated with an increased risk of myocardialinfarction, and recently it was demonstrated that the associationbetween β-carotene and acute myocardial infarction depends on thepolyunsaturated fatty acids status, and that feeding the all-transisomer of β-carotene to cholesterol-fed rabbits attenuated the extent oftheir atherosclerosis, with no effect on LDL oxidizability ex vivo. Dataon the ability of β-carotene supplementation in vitro or in vivo toprotect LDL from oxidation are conflicting: some studies found aninhibitory effect of β-carotene supplementation on LDL oxidation,whereas several other did not find such effect.

Lycopene, the open chain analog of β-carotene, shares with it similarstructure with an extended conjugated double bonds. In human plasma,lycopene and β-carotene are quantitatively the major carotenoids.Lycopene was shown to possess the greatest quenching ability of singletoxygen among the various carotenoids (DiMascio P., Kaiser S. & Sies H.,“Lycopene as the most efficient biological carotenoid singlet oxygenquencher,” Arch. Biochem. Biophys., 1989, 274: 532-538) and it was shownto be at least twice as effective antioxidant as β-carotene inprotecting blood lymphocytes form NO₂ radical damage (Bohm F., TinklerJ. H., and Truscott T. G., “Carotenoids protect against cell membranedamage by the nitrogen dioxide radical”, Nature Medicine, 1995, 2:98-99).

We have recently demonstrated a protective effect of tomatoes lycopeneagainst oxidative modification of LDL. This protection of LDL bylycopene exceeds the protection exhibited by β-carotene, was selectiveonly to LDL's with high vitamin E content and was shown when thecarotene was present in combination with vitamin E. (see e.g., FuhrmanB, Ben Yaish L, Attias J. Hayek T. Aviram M. Tomatoes lycopene andβ-carotene inhibit low density lipoprotein oxidation and this effectdepends on the lipoprotein vitamin E. content. Nutr Metab. Cardiovasc.Dis 7: 433-443, 1997). Furthermore, we have also demonstrated thatdietary supplementation of lycopene acts as moderate hypocholesterolemicagent secondary to its inhibitory effect on cellular cholesterolsyntheses (see e.g. Fuhrman B., Elis A., Aviram M., Hypocholesterolemiceffect of lycopene and β-carotene is related to suppression ofcholesterol synthesis and augmentation of LDL receptor activity inmacrophages. Bichem. Biophys. Res. Commun. 233: (658-662, 1997).

Israel Patent Application No. 123,132 recently filed covers the use of amixture of lycopene and garlic to be used in preventing LDL oxidation.

OBJECTIVES

It is an objective of the present invention to provide a novel mixtureof lycopene and vitamin E. It is an objective of the present inventionto provide a synergistic mixture of lycopene and vitamin E active inblocking the oxidation of LDL and/or atherosclerosis and reduce thecholesterol levels in plasma. An additional objective of the presentinvention is the use of the above described mixture to prepare apharmaceutical or dietary composition for arresting the progression ofatherosclerosis. A further objective of the present invention is toprovide a composition for the arresting of the progression ofatherosclerosis containing essentially products acceptable anddesertable of the harm diet.

SUMMARY OF THE INVENTION

The invention is based on the discovery that there is an unexpected andsurprising synergistic effect between lycopene and vitamin E, in theprevention of LDL oxidation. Lycopene is a natural product and ispresent in tomatoes in various concentrations, but can also be producedby fungal, algal or by fermentation, in genetically modified organisms(GMO) or by synthetic method. Compositions comprising it and their useare comprised in the present invention. A widely used source of vitaminE is soybean distillate.

The invention therefore provides a composition which is a combination oflycopene and vitamin E.

The composition may contain other dietary components, additives,excipients, binding agents, coatings, etc., or other compounds that haveno significant effect in preventing LDL oxidation. The compositionsaccording to the invention, in view of their purpose, are pharmaceuticalcompositions. However, since their components are individuallyacceptable ingredients of the human diet, and in fact are individuallypresent in foodstuffs, they may be considered and used as dietary orhealth supplements.

The use of the aforesaid combinations of compounds as medicines orcomponents of pharmaceutical compositions or of dietary or healthcompositions for the prevention of LDL oxidation is also an aspect ofthe present invention.

The use of tomato oleoresin for the prevention of LDL oxidation is aspecific aspect of the present invention.

The compositions according to the invention should preferably compriselycopene and vitamin E in a molar proportion of 1:400 to 10:1,preferably 1:80 to 5:1. The amount of lycopene in each unit of thecomposition, e.g. a tablet or soft gel capsule, should be in the rangeof 1-4, preferably about 2-3 and up to 15 mg of lycopene.

The invention comprises a method of preventing LDL oxidation, andtherefore arresting the progression of atherosclerosis, which comprisesadministering to a patient lycopene and vitamin E, in the aforesaidrelative amounts.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In a preferred way of carrying out the invention, and in the Examplesthat will be described, lycopene was obtained from tomato oleoresin. Theoleoresin was supplied by LycoRed-Natural Products Industries, Ltd, BeerSheva, Israel. It consisted of crystalline lycopene (6.0%) suspended inthe lipid phase of the tomato. Fatty acids as triglycerides constitutedthe major part (72%) of the oleoresin, whereas 19% were non-saponifiedmaterials including 6.0% lycopene, 0.1% β-carotene and 1% vitamin E. Therest consists of water and water soluble materials. The lycopene usedwas extracted and purified mainly in the all-trans configuration.Lycopene can be extracted from tomato oleoresin, as described in PatentApplication No. WO 97/48287.

Vitamin E is readily available in the pharmaceutical market.

Said ingredients are conveniently available and used in the form ofoleoresin or in a solid form such as beadlets.

Materials

Supplementation of Human LDL with Lycopene

Stock solutions of 1 mM of purified lycopene or of 10 mg/ml of tomato'soleoresin, were prepared in Tetrahydrofuran (THF, HPLC grade). Allprocedures were performed in dim light. LDL was incubated with therespective concentrations of tomato's oleoresin or with 3 μM of lycopene(3 μl/ml THF, derived from a stock solution of 1 mM) for 30 minutes at37° C. in the dark. LDL incubated with THF alone served as control.

LDL Oxidation

Oxidation of LDL was carried out in a shaking water bath at 37° C. underair, in plastic tubes (1 cm in diameter). For metal ions dependentoxidation LDL was incubated for 4 hours at 37° C. with freshly preparedCuSO₄ (5 μM).

LDL oxidation was determined by measuring the amount of thiobarbituricacid reactive substances (TBARS) (See: Buege J. A. and Aust S. D.Microsomal lipid peroxidation. Methods Enzymol. 1978; 52: 302-310) andby lipid peroxides formation using a commercially available kit.

Effects of Lycopene or Vitamin E Alone Against LDL Oxidation

LDL (100 μg of protein/ml) was preincubated for 30 min at 37° C. witheither vitamin E or lycopene in various forms. LDL oxidation was inducedby the addition of 5 μM of CuSO₄ and was preincubated for 30 min at 37°C. LDL oxidaton was measured by the TBARS assay or by measuring theinhibition of the formation of lipid peroxides. Results are shown inFIG. 1 for vitamin E inhibition and FIG. 2 for crystalline lycopene.FIG. 3 shows the effect for tomato oleoresin, and for comparison, FIG. 4shows the results for tomato oleoresin where the natural occurringvitamin E was extracted out.

Effect of Lycopene in Combination With Vitamin E Against LDL Oxidation

The antioxidative effect of a combination of vitamin E with lycopeneagainst LDL oxidation, was assessed is a manner illustrated by FIGS. 5to 7, in which the degree of inhibition of LDL oxidation by individualanti-oxidants is shown in the ordinate.

The tests hereinbefore described show that when vitamin E and lycopeneare added together to LDL during copper-induced oxidation, a synergisticeffect occurs. This synergistic inhibitory effect on LDL oxidation, isbelieved to be probably related to the contribution of vitamin E andlycopene to different antioxidative mechanisms. Whereas vitamin E is achain breaking agent and acts as a free radicals scavenger, lycopene isa potent free radical scavenger as well as an oxygen quencher.Therefore, administration of a combination of these two antioxidantswith synergistic inhibitory effects on LDL oxidation, such as vitamin Eand lycopene, appears to be beneficial over the administration ofindividual antioxidants separately. This is further evidenced by theinhibitory effect on LDL oxidation of tomato's oleoresin, which containsa combination of antioxidants.

In some cases as published in the literature, vitamin E alone does notefficiently lower the LDL oxidation. Nevertheless, when lycopene or theoleoresin of tomatoes is added to the vitamin E, a marked synergisticlowering of the LDL oxidation occurs.

EXAMPLES

General

FIG. 1: Vitamin E dose dependent inhibition Of CuSO₄-induced LDLoxidation as measured by TBARS (A) or lipid peroxide (B) formation.Vitamin E is dissolved in tetrahydrofuran and the IC₅₀ for theinhibition of TBARS and lipid peroxides formation is 6 μM and 6.5 μM,respectively.

FIG. 2: Crystalline lycopene inhibition of copper-ions induced LDLoxidation. The maximal concentration used (50 μM) did not achieve 50%inhibition, neither of TBARS nor of lipid peroxides formation.

FIG. 3: LYC-O-MATO (6%) (where LYC-O-MATO is Lycored Natural ProductsIndustries Ltd trade name for natural tomato oleoresin) inhibition ofcopper ions-induced LDL oxidation. The IC₅₀ for the inhibition of TBARSis 7.8 μM. The maximal concentration used (12 μM did not achieve 50%inhibition of lipid peroxides formation.

FIG. 4: Inhibition of copper ions-induced LDL oxidation of LYC-O-MATO(6%), which does not contain vitamin E {the vitamin E was extracted outand the material is designated as LYC-O-MATO (DW)}. The IC₅₀ for theinhibition of TBARS is 7.8 μM. The maximal concentration used (12 μM didnot achieve 50% inhibition of lipid peroxides formation

FIG. 5: Shows the results of Table 1.

FIG. 6: Shows the synergistic effect of vitamin E and LYC-O-MATO.

FIG. 7: Shows the synergistic effect of vitamin E and vitamin Eextracted LYC-O-MATO.

The 1% naturally occurring vitamin E concentrates of tomato oleoresin isequal to 0.213 μM vitamin E where said oleoresin contains 6% lycopene.Thus the true concentration of vitamin E in FIG. 6 is what is listedplus 0.213 μM. FIG. 5 gives data using crystalline lycopene which doesnot contain any vitamin E. Similarly, for FIG. 7 which used tomatooleoresin where the vitamin E and other waxes were removed.

Example 1

The Synergistic effects of a combination of vitamin E and crystallinelycopene on copper ions-induced LDL oxidation was studied. Details areshown in Table 1, affording the individual components of the ColbyEquation used to calculate synergism as follows:

A+B−(AB/100)=Expected Control

where

A=Percent control of vitamin E alone

B=Percent control of lycopene alone

and

synergism=I _(F) /I _(E)

where

I_(F)=Actual Control in Percent

I_(E)=Expected Control in Percent

and synergism is proven if the ratio of I_(F)/I_(E) is significantlygreater than 1. The results are shown in Table 1 and FIG. 5.

Example 2

Following the method of Example 1, the synergistic effect of vitamin Eand LYC-O-MATO (lycopene 6%) were determined. Results are shown in Table1 and FIG. 6.

Example 3

Following the method of Example 1, the synergistic effect of vitamin Eand vitamin E extracted LYC-O-MATO (lycopene 6%) were determined.Results are shown in Table 1 and FIG. 7.

TABLE 1 DATA (%) FROM EXPERIMERNTS 1-3 FITTED TO THE COLBY EQUATION.Exper- I_(F) I_(F) I_(F)/I_(E) I_(F)/I_(E) iment ^(a)A ^(a)B I_(E)(TBARS) (LIPIDS) (TBARS) (LIPIDS) ^(b)1 0  0   0  36 18 infiniteinfinite ^(c)2 0 ^(d)42 ^(d)42 68 49 1.62 1.53 ^(e)32 ^(e)32 ^(f)3 0^(d)12 ^(d)12 45 25 3.75 infinite ^(e)0  ^(e)0  ^(a)A = vitamin E; ^(a)B= lycopene. ^(b)1 μM A & 1 μM B ^(c)1 μM A & 6 μM B ^(d)inhibition ofTBARS ^(e)inhibition of lipid peroxides ^(f)1 μM A & 5 μM B

While embodiments of the invention and experimental data have beendescribed by way of illustration, it should be understood that theinvention is not limited thereto and may be carried out with manymodifications, vairations and adaptations, without departing from itsspirit or exceeding the scope of the claims

What is claimed is:
 1. A synergistic pharmaceutical or dietarycomposition which comprises synergistic effective amounts of lycopeneand vitamin E.
 2. A composition in accordance with claim 1 wherein thelycopene is selected from the group consisting of lycopene from tomatooleoresin, algal, fermented, fungal, genetically modified organisms(GMO), synthetic lycopene and mixtures thereof.
 3. A composition inaccordance with claim 1 wherein the concentration of vitamin E is in therange of 0.025 μM to 50 μM.
 4. A composition in accordance with claim 1wherein the concentration of lycopene is in the range of 0.025 μM to 50μM.
 5. A composition in accordance with claim 3 wherein the molar ratioof lycopene to vitamin E is 1:400 to 10:1.
 6. A composition inaccordance with claim 1 wherein the composition contains a compoundselected from the group consisting of dietary components, additives,excipients, binding agents, coatings, preservatives and mixturesthereof.
 7. A composition in accordance with claim 1 wherein thelycopene and vitamin E mixture is contained in a dosage form selectedfrom the group consisting of tablets, caplets, vegecaps and hard shellgelatin capsules.
 8. A method for arresting the progression ofatherosclerosis comprising synergistic effective amounts ofadministering to a subject in need thereof a synergistic compositioncontaining lycopene and vitamin E.
 9. A method in accordance with claim8 wherein the lycopene is selected from the group consisting of lycopenefrom tomato, algal, fermented, fungal, genetically modified organisms(GMO), synthetic lycopene and mixtures thereof.
 10. A method inaccordance with claim 8 wherein the vitamin E concentration is in therange of 0.025 μM to 50 μM.
 11. A method in accordance with claim 8wherein the concentration of lycopene is in the range of 0.025 μM to 50μM.
 12. A method in accordance with claim 8 wherein the molar ratio oflycopene to vitamin E is 1:400 to 10:1.
 13. A method in accordance withclaim 8 wherein the composition contains a compound selected from thegroup consisting of dietary components, additives, excipients, bindingagents, coatings, preservatives and mixtures thereof.
 14. A method inaccordance with claim 8 wherein the lycopene and vitamin E mixture iscontained in a dosage form selected from the group consisting oftablets, caplets, vegecaps and hard shell gelatin capsules.
 15. A methodfor blocking the oxidation of LDL in serum which comprises synergisticeffective amounts of contacting the serum with a synergistic compositionwhich comprises lycopene and vitamin E.
 16. A method in accordance withclaim 15 wherein the lycopene is selected from the group consisting oflycopene from tomato, algal, fermented, fungal, genetically modifiedorganisms (GMO), synthetic lycopene and mixtures thereof.
 17. A methodin accordance with claim 15 wherein vitamin E concentration is in therange of 0.025 μM to 50 μM.
 18. A method in accordance with claim 15wherein the concentration of lycopene is in the range of 0.025 μM to 50μM and most 0.5 μM.
 19. A method in accordance with claim 17 wherein themolar ratio of lycopene to vitamin E is 1:400 to 10:1.
 20. A method inaccordance with claim 15 wherein the composition contains a compoundselected from the group consisting of dietary components, additives,excipients, binding agents, coatings, preservatives, and mixturesthereof.
 21. A method in accordance with claim 15 wherein the lycopeneand vitamin E mixture is contained in a dosage form selected from thegroup consisting of tablets, caplets, vegecaps and hard shell gelatincapsules.
 22. The method of claim 8 wherein the vitamin E concentrationis 0.5 μM to 5 μM, the concentration of lycopene is 0.5 μM to 10 μM, andthe molar ratio of lycopene to vitamin E is 1:80 to 5:1.